Isolation and Characterization of a Bacterium Able to Degrade High Concentration of Iprodione
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Canadian Journal of Microbiology Isolation and characterization of a bacterium able to degrade high concentration of iprodione Journal: Canadian Journal of Microbiology Manuscript ID cjm-2017-0185.R1 Manuscript Type: Article Date Submitted by the Author: 30-Sep-2017 Complete List of Authors: Cao, Li; Hexi University Shi, Wenhong; Hexi University Shu, Rundong; Hexi University Pang, Jian;Draft Hexi University Liu, Yuetao; Hexi University Zhang, Xiaohua; Hexi University Lei, Yuming; Hexi University, College of Agriculture and Biotechnology Is the invited manuscript for consideration in a Special N/A Issue? : Keyword: Biodegradation, Iprodione, Metabolite, Microbacterium https://mc06.manuscriptcentral.com/cjm-pubs Page 1 of 22 Canadian Journal of Microbiology Isolation and characterization of a bacterium able to degrade high concentration of iprodione Li Cao, Wenhong Shi, Rundong Shu, Jian Pang, Yuetao Liu, Xiaohua Zhang, Yuming Lei * College of Agriculture and Biotechnology , Hexi University, Zhangye , Gansu , 734000, China Draft *Corresponding author. Tel.: +86-936-8281523. E-mail address: [email protected] 1 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 2 of 22 Abstract: A bacterial strain CQH-1 capable of mineralizing iprodione was isolated and characterized. In combination with morphological, physiological & biochemical characters, and the phylogenetic analysis of 16S rRNA gene sequence, strain CQH-1 was identified as Microbacterium sp.. It could use iprodione and 3,5-dichloroaniline as the sole carbon source and energy source for growth. It completely degraded 100 mg L -1 of iprodione within 96 h at 30 °C. During the degradation of iprodione by strain CQH-1, two compounds were detected in GC-MS analysis and recognized as N-(3,5-dichlorophenyl)-2,4-dioxoimidazolidine and 3,5-dichloroaniline, respectively. So, the biodegradation pathway of iprodione by strain CQH-1 was proposed. This is the first report of iprodione-mineralizing strain from the genus of Microbacterium , and strain CQH-1 might be a promising candidate for its application in the bioremediation of iprodione-contaminated environments. Draft Key words: Biodegradation; Iprodione; Metabolite; Microbacterium 1. Introduction Iprodione [3-(3,5-dichlorophenyl)N-isopropyl-2,4-dioxoimidazolidine-1-carboxamide] is a dicarboxamide fungicide that inhibits DNA and RNA synthesis, cell division and cellular metabolism in fungi (Davidse. 1986), which is commonly employed in a variety of greenhouse and field crops to control fungal infestations by Botrytis cinerea , Alternaria sp., Monilinia fructigena , Rhizoctonia solani , Sclerotinia sclerotiorum , Aspergillus sp., Penicillium sp., Sclerotinia sp. and other fungal pathogens (Miñambres et al. 2010; Wang et al. 2012; Morales et al. 2013; Grabke et al. 2014; Campos et al. 2015). Interestingly, benzimidazole resistance is now widespread because of its frequent and indiscriminant use, while iprodione was developed in response to resistance problems of benzimidazoles 2 https://mc06.manuscriptcentral.com/cjm-pubs Page 3 of 22 Canadian Journal of Microbiology (Sequinatto et al. 2013). Iprodione is moderately persistent in soil with a half-life time of 7–60 d depending on the environmental conditions (Carmona et al. 2001). It is relatively mobile in the soil environment and leachable into groundwater and surface water, so its residues have been detected in many environment samples (Derbalah et al. 2003; Omirou et al. 2009; Wang et al. 2012; Sequinatto et al. 2013; Allen et al. 2015). Iprodione has been classified as a probable carcinogen, because iprodione shows relatively high toxicity to crustaceans, moderately toxicity to fish and slight toxicity to birds (Morales et al. 2013). It could delay male rat pubertal development, reduce serum testosterone levels, decrease ex vivo testicular testosterone production, and even have potential oncogenic effect (Washington and Tchounwou, 2004; Blystone et al. 2007). There are also a few reports that iprodione may inhibit the microbes of environment and have an impact on the diversity of soil microbialDraft communities (Wang et al. 2004; Verdenelli et al. 2012). So, the presence of iprodione residues is a matter of serious concern. Microbial biodegradation is the main mechanism responsible for the dissipation of iprodione. To date, several bacterial strains capable of degrading iprodione have been reported, including Arthrobacter sp. strain MA6 (Athiel et al. 1995), four combined Pseudomonas strains (Mercadier et al. 1997) and Arthrobacter sp. strain C1 (Campos et al. 2015). During the degradation of iprodione, N-(3,5-dichlorophenyl)-2,4-dioxoimidazolidine, 3,5-dichlorophenylurea acetate and 3,5-dichloroaniline were the most frequently detected intermediate metabolites (Athiel et al. 1995; Mercadier et al. 1997; Campos et al. 2017), and then the biodegradation pathway of iprodione was proposed. Bioremediation has received increasing attention as a reliable, effective, safe, cost-effective and promising approach to clean up polluted environments (Rajendran et al. 2003; Chen et al. 2011; Cao et al. 2013). Several pesticides including parathion, atrazine, and chlorpyrifos have been successfully removed from soil and aquatic environments using microbes (Munnecke and Hsieh 1974; 3 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 4 of 22 Singh and Walker 2006; Yadav 2014; Zhang 2015). However, no report has been made on bioremediation of iprodione. The objective of the present study was to isolate a bacterium able to degrade high concentration of iprodione, to study the effects of pH and temperature on biodegradation of iprodione, to analyze the metabolites appeared during the degradation of iprodione, and finally to provide a candidate for the bioremediation of iprodione contaminated environments. 2. Materials and methods 2.1. Chemicals and media Iprodione and 3,5-dichloroaniline (analytical standards: >97% purity) were purchased from Sigma-Aldrich of USA. DNA polymerase, T4 ligase, DNA gel extraction kit, plasmid extraction kit for DNA manipulationDraft were purchased from Takara Biotechnology Co. Ltd. (Dalian, China). Other conventional reagents used in this study were of the highest analytical-reagent grade. -1 The mineral salts medium (MSM) contains (g L ): K 2HPO 4 1.5, KH 2PO 4 0.5, MgCl 2 0.2, NaCl 1.0, NH 4NO 3 1.0 , pH 7.0. Iprodione or 3,5-dichloroaniline was used as the sole carbon source. Concentrated stock solution of iprodione or 3,5-dichloroaniline (10 g L -1) was prepared in acetone solution, which was sterilized via membrane filtration and diluted into sterile flask to achieve the desired concentrations, then the corresponding volume of medium was poured into the flasks after acetone has volatilized. Luria–Bertani (LB) medium contains (g L -1): tryptone 10.0, NaCl 10.0 and yeast extract 5.0, pH 7.0. Solid medium were prepared by adding 15 g L -1 agar into above liquid media. 2.2. Isolation and identification of iprodione-degrading bacteria Iprodione-degrading bacteria were isolated via the enrichment culture technique. The samples were collected from the soil of corn field located in the south of Zhangye in Gansu 4 https://mc06.manuscriptcentral.com/cjm-pubs Page 5 of 22 Canadian Journal of Microbiology province of China (38°3216′′ N and 99°25′38′′ E) with previous exposure to iprodione. The 10 g of sample was added into a 250 mL flask with 100 mL of sterile MSM containing 100 mg L-1 iprodione and incubated on a rotary shaker (160 rpm) at 30 °C for 7 days. The suspension (5 mL) was transferred to fresh MSM containing 100 mg L -1 iprodione and incubated for another 7 days. After five rounds of enrichment, the culture was diluted and spread onto solid MSM plates containing 100 mg L -1 iprodione and incubated at 30 °C for 5 days. Iprodione-degrading ability was observed in terms of the formation of transparent halos surrounding the colonies. A bacterial strain designated as CQH-1 that formed transparent halos around their colonies was selected for further study. Strain CQH-1 was identified according to Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994). Its 16S rRNA gene was amplified via polymerase chain reaction (PCR) using standard proceduresDraft (Lane, 1991). The PCR product was purified, ligated into pMD19-T, and transformed into E. coli DH5α. An automatic sequencer was used to determine the 16S rRNA gene sequence. The 16S rRNA gene sequence was deposited at GenBank (Accession No. KX618653). Multiple alignment analysis of different 16S rRNA gene sequences in the GenBank database was performed using ClustalW 1.8.3 with default settings. The sequence alignments were analyzed using MEGA5 software. The distances were calculated using the Kimura two-parameter distance model. Unrooted phylogenetic trees were constructed using the neighbor-joining method. Datasets were bootstrapped 1000 times (Saitou and Nei, 1987). 2.3. Biodegradation of iprodione in liquid-culture condition Strains were collected by centrifugation at 6000×g for 5 min at room temperature after precultured for 16 h in LB medium at 30 °C on a shaker at 160 rpm. The cell pellets were washed twice with MSM and adjusted to an optical density of 1.0 at 600 nm. Unless otherwise stated, the cells were inoculated at 3% (v/v) into a 250 mL flask containing 100 mL 5 https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 6 of 22 of MSM at pH 7.0 amended with 100 mg L-1 iprodione or 30 mg L -1 3,5-dichloroaniline as sole carbon and energy sources and incubated at 30 °C and 160 rpm in a rotary shaker. At each sampling point, the samples were used to determine the concentration of iprodione, the concentration of 3,5-dichloroaniline, and cell density (OD 600 nm). Uninoculated medium was used as the control. All experiments were replicated for three times. 2.4. Effect of pH and temperature on biodegradation of iprodione The MSM at different pH (4.0-8.0) containing iprodione (100 mg L -1) were used to study the effect of pH on degradation rate of iprodione at 30 °C.